Experimentally testing mate preference in an avian system with unidirectional bill color introgression

Abstract Mating behavior can play a key role in speciation by inhibiting or facilitating gene flow between closely related taxa. Hybrid zones facilitate a direct examination of mating behavior and the traits involved in establishing species barriers. The long‐tailed finch (Poephila acuticauda) has two hybridizing subspecies that differ in bill color (red and yellow), and the yellow bill phenotype appears to have introgressed ~350 km eastward following secondary contact. To examine the role of mate choice on bill color introgression, we performed behavioral assays using natural and manipulated bill colors. We found an assortative female mating preference for males of their own subspecies when bill color was not manipulated. However, we did not find this assortative preference in trials based on artificially manipulated bill color. This could suggest that assortative preference is not fixed entirely on bill color and instead may be based on a different trait (e.g., song) or a combination of traits, or alternatively may be due to lower statistical power alongside the bill manipulations being unconvincing to the female choosers. Intriguingly, we find a bias in the inheritance of bill color in captive bred F1 hybrid females. Previous modeling suggests that assortative mate preference and this kind of partial dominance in the underlying genes may together contribute to introgression, making the genetic architecture of bill color in this system a priority for future research.

and preferences for them, can lead to assortative mating between lineages, such outcomes are not guaranteed (Ritchie, 2007). For example, if the forces of selection by which a novel trait spreads to fixation within one population are shared among populations then sexual selection will act against population divergence (Miñano et al., 2021). Indeed, the traits that distinguish hybridizing lineages appear to move across hybrid zones with an appreciable frequency (Baldassarre et al., 2014;Lipshutz et al., 2019;Parsons et al., 1993;While et al., 2015). Experimental evaluation of the strength and targets of sexual selection is, therefore, a key component for understanding the evolution of reproductive isolation and patterns of introgression in hybrid systems.
Hybridization can facilitate the sharing of beneficial genetic mutations and phenotypes that accumulated in parental lineages during periods of isolation (Abbott et al., 2013;Harrison, 1986;Marques et al., 2019;Wu, 2001). The extent of introgression will be influenced by the fitness implications of these novel gene combinations and resulting phenotypes, together with the local recombination rate, epistatic interactions, and demography (Barton & Bengtsson, 1986;Schumer et al., 2018;Wu, 2001). Traits that reduce fitness in the receiving lineage will show resistance to introgression, and act to limit the introgression of linked genetic material, whereas unlinked selectively neutral alleles may flow unopposed between hybridizing lineages (Barton, 1979;Gompert et al., 2012;Harrison, 1990;Payseur, 2010). In contrast, theory predicts that strong positive selection can drive the introgression of adaptive genotypes and phenotypes between lineages (Baldassarre et al., 2014;Barton, 2001; Hedrick, 2013; Morjan & Rieseberg, 2004). Indeed, most cases of unidirectional introgression of color traits that have been studied in detail have concluded that they were likely adaptive, resulting from fitness benefits due to aposematism or sexual selection (Baldassarre et al., 2014;MacGregor et al., 2016;Pardo-Diaz et al., 2012;Stein & Uy, 2006;While et al., 2015).
Mate choice could contribute to unidirectional introgression of phenotypic traits through two main mechanisms. Firstly, if a novel trait emerges in one population but a preference for it exists across all populations, we would expect selection to result in the introgression of this trait between populations following secondary contact, so long as the allelic variation for the novel trait is not incompatible with standing variation in recipient populations (Wirtz, 1999). This phenomenon appears to be driving introgression of the golden collars from golden collared manakins into the white-collared species (Stein & Uy, 2006), and the introgression of red back plumage into orange-backed subspecies in the red-backed fairy wren (Baldassarre et al., 2014;Baldassarre & Webster, 2013). Secondly, and somewhat counterintuitively, two recent studies of avian hybrid zones employed simulations to reveal that assortative mating can also result in unidirectional introgression, when combined with epistasis or dominance among underlying genes (Metzler et al., 2021;Semenov et al., 2021). These cases demonstrate that mate choice can be important for introgression, but this remains relatively understudied, so additional data on species in contact will provide a broader understanding of the evolutionary forces underlying unidirectional introgression.
The long-tailed finch Poephila acuticauda is an Australian estrildid finch characterized by a yellow-billed subspecies in the West (P. a. acuticauda) and a red-billed subspecies in the East (P. a. hecki) that come into secondary contact on the eastern edge of the Kimberley Plateau (Figure 1a, Hooper et al., 2019). In addition to their difference in bill color, subspecies are also distinct in their nuclear and mitochondrial genomes (Hooper et al., 2019;Lopez et al., 2021), and to some degree in their songs (Zann, 1976) and sperm size (Rowe et al., 2015). The respective geographic centers of genetic and bill color admixture are significantly displaced from each other: the hybrid zone for bill color (as well as three candidate genes underlying variation in this trait) is located 350 km east of the hybrid zone for the vast majority of the genome (Hooper et al., 2019;Lopez et al., 2021). Genetic analyses suggest that variation for yellower bills has introgressed from subspecies acuticauda into subspecies hecki (Hooper et al., 2019;Lopez et al., 2021). Given that bill color in the long-tailed finch is an oligogenic trait-that is, heritable variation is seemingly governed by only a small number of genes- (Hooper et al., 2019) and has a steep phenotypic cline, it is unlikely that stochasticity alone is responsible for this apparent unidirectional introgression of bill color. Instead, these findings suggest that introgression of the yellow billed phenotype may have an adaptive basis and so increase fitness in some way (Barton, 2001;Harrison & Larson, 2016). We hypothesize that precopulatory matechoice may increase fitness associated with yellow bill color and thus may be responsible for the unidirectional introgression observed in the long-tailed finch, as observed in other birds (Baldassarre & Webster, 2013;Stein & Uy, 2006). There is little variation in bill color within allopatric populations (Griffith & Hooper, 2017), and a previous study that assessed assortative mating in one such allopatric population with yellow bills found no evidence of preference for the limited within-population variation in bill coloration (van Rooij & Griffith, 2012). Here, we are not looking at sexual selection for an ornamental trait that varies significantly across the males within a single population (i.e. Gustafsson et al., 1995), but rather considering selection on a trait (red vs. yellow bills) that varies across a (sub-) species boundary, that is, a species indicator trait (Saetre et al., 1997).
Here, we describe behavioral trials conducted on wild-caught and first-generation captive long-tailed finches of both subspecies.
Both natural and experimentally manipulated bill colors were used.
Given that the genetic architecture of traits (e.g., dominance and epistasis) can play an important role in trait inheritance and the dynamics of introgression following hybridization, we also examined bill color of captive-bred first generation (F 1 ) hybrids to gain insight into the possible role of these processes in the long-tailed finch system.

| Study animals
Long-tailed finches used in these behavioral trials were either wildcaught or the first-generation pure-bred offspring of wild-caught  Given subsequent findings about the long-tailed finch hybrid zone, particularly the apparent introgression of color (Hooper et al., 2019;Lopez et al., 2021), we revisit and analyze these data here to assess the role mate choice might have in bill color introgression.

| Bill color manipulations
To experimentally determine whether any observed preference between subspecies is due to differences in bill color, for some trials the stimulus bird bill colors were manipulated using nail polish to be F I G U R E 1 Bill color differentiation between subspecies of the long-tailed finch (Poephila acuticauda). (a) Approximate geographic distribution of bill color variation across the range of the long-tailed finch from yellow bills in the west, red bills in the east, and a narrow region of orange bills in between following from Griffith and Hooper (2017). The location of primary genomic admixture (Z chromosome and mtDNA) between subspecies is represented as a dashed blue line following from Hooper et al. (2019) and Lopez et al. (2021) with arrows representing the hypothesized direction of introgression of yellow bill color from west to east. The location of source populations of birds used in this study are represented on the map as black circles (see Methods for precise locations). (b) Representative photograph of long-tailed finches from our research colony used in this study: from left, subspecies acuticauda, subspecies hecki. (c) Standardized reflectance spectra for bills from individuals of subspecies acuticauda (N = 71) and hecki (N = 80), as well as red (N = 5) and yellow (N = 5) artificial bill manipulations, are shown as mean and standard error. The mean and standard deviation for bill hue (colorimetric variable H3; the wavelength midway between maximum and minimum reflectance between 400 and 700 nm) is shown for each subspecies as vertical lines. (d) Natural variation in bill color hue (H3) for individuals of each subspecies compared against our artificially manipulated yellow and red treatments. Our artificial yellow manipulation overlaps the range of variation observed in natural acuticauda bills but our artificial red manipulation is redder than the range of variation observed in natural hecki bills. Different letters indicate statistical significance between treatment groups (p < 0.05; Tukey's HSD test). . These red and yellow nail polish colors were selected as they appeared to be the closest match to the bill colors of each subspecies by the human eye. To assess how effective our bill manipulations were we used reflectance spectrophotometry to measure bill color of both unmanipulated individuals and individuals with manipulated bills, following published protocols (Griffith & Hooper, 2017;McDiarmid et al., 2022;van Rooij & Griffith, 2012). Spectral reflectance of the center of the upper mandible was recorded three times using a USB2000 + Miniature Fiber Optic spectrophotometer curve (e.g., Sheldon et al., 1999). Similarly here the natural bill color contains some variation in the UV part of the spectrum between subspecies that we were unable to capture with our manipulations (see Figure 1c). As such our manipulations are principally limited to the visual spectrum. As animals integrate visual signals from the visual (400-700 nm) and UV (300-400 nm) parts of the spectrum together in natural signals (Vorobyev & Osorio, 1998), experimental manipulations can still be effective for the purposes of testing the role of color signals as long as any noise in the UV signal does not overwhelm that in the visual part of the spectrum. To examine whether our metric H3 was capturing variation across both the UV and visual spectrums, we performed a principal component analysis (PCA) of the smoothed and normalized spectral data (as above) in both the UV and visual spectrum (300-700 nm) from natural and manipulated red and yellow birds (4 of each group, total 16 individuals). The first two principal components (PC1 and PC2) accounted for over 80% of total variance ( Figure A4, Table A4), so we then examined whether variation in H3 was accounted for by PC1 and PC2 (by running a linear model with H3 as the response variable and PC1 and PC2 as fixed effects). PC1 and PC2 explained a large amount of the variation described in H3 (R 2 = 0.83). This suggests that our use of H3 is capturing the bulk of the main spectral variation and should therefore appropriately represent the yellow and red phenotypes.
The 'black' manipulation was intended to mimic the coloration of juveniles, and the manipulation successfully resulted in a uniform and low reflectance across the avian color spectrum. There was no significant difference between the hues of bills prior to and following treatment with our clear control manipulation (paired t test: t = 1.5, p = 0.2385, n = 4).

| Four-way mate-choice trials
The first set of trials performed exposed a focal (choosing) bird to four stimulus birds, all with manipulated bill colors. We describe the methodology and results of these 'four-way trials' for completeness and transparency, however, they have issues (outlined below) that limit the interpretation of these trials. A second set of trials ('twoway trials') with refined methodology were subsequently performed and we will focus on those.
The four-way mate choice trials were performed in a four-armed wooden preference apparatus with the focal (choosing) bird starting on a perch in the middle ( Figure A1). Each arm contained a perch in front of Plexiglass (Perspex; Plexiglas®, Röhm, Germany) following (Pryke & Griffith, 2007), behind which there was a stimulus bird of the opposite sex to the focal bird. These trials were performed with both male and female focal birds. All four stimulus birds had bill color manipulations (as described above), with each trial including one each of (1) yellow, (2) red, (3) black, or (4)

| Two-way female mate-choice trials
The second set of trials (two-way trials) were performed using only female focal birds, who were provided just two stimulus males at a time, with separate trials for manipulated and unmanipulated stimulus bill colors and an increased overall sample size. For these two-way trials the existing four-way testing apparatus was modified by blocking off two opposite arms ( Figure 2). We wanted to focus on one sex to improve our sample sizes and in birds females are typically considered the choosier sex (Andersson, 1994;Andersson & Simmons, 2006), and in our four-way trials there was no evidence of preference between red and yellow bills shown by males (Supporting Information; Table S3). In all two-way trials, focal females were presented with one stimulus male with a yellow bill and one with a red bill. Focal females in 'natural' trials were presented with a choice between two males, one male of each subspecies with their natural bill colors. In order to control for other traits that might differ between subspecies (e.g., song), or to account for the differing reactions of a consubspecific male to a focal female, a separate set of trials were run in which focal females were presented with a choice between two males of their own subspecies (i.e., consubspecific males), however, both males had their bill color manipulated by nail polish, to be either artificial yellow or artificial red. We focused analyses on the proportion of time the focal bird spent in front of each stimulus bird, calculated as a proportion of the total time spent in front of any stimulus birds, because the absolute time could be skewed by certain trials with large absolute values. Nonetheless, because a longer absolute length of time spent may reflect increased preference, we also ran analyses using the absolute time and find similar results (Table A2).
We performed a total of 87 trials with stimulus birds having natural bill colors, 41 with an acuticauda female and 46 with a hecki female, each female given a single trial. Of these, 70 were successful (i.e., in 17 cases the focal female did not move for 15 min). Of the successful trials, 32 were with acuticauda females (64 different males were used, each used once) and 38 were with hecki females (70 different males used either once or twice; mean = 1.08, SD = 0.28). For the trials with artificial bill colors for the stimulus birds, fewer were performed due to the challenges associated with bill color manipulation. A total of 40 trials were performed using 16 hecki females and 24 acuticauda females. Of these, 36 were successful (i.e., in 4 cases the focal female did not move for 15 min). Of the successful trials, 13 had acuticauda females (11 different stimulus males were used) and 23 had hecki females (10 stimulus males were used).

| Bill color inheritance in F 1 hybrids
To evaluate evidence of bill color inheritance in hybrids, we measured the bill color of 110 captive bred first-generation (F 1 ) hybrids via reflectance spectrophotometry using the methods outlined above.
F 1 hybrids were produced by establishing mixed breeding pairs containing a male from a parental subspecies with a female from the other parental subspecies in small aviaries at Macquarie University.
As one candidate locus for bill color is located on the Z chromo-

| Statistical methods
For statistical analyses we used R version 4.1.2 (R Core Team, 2020),  (Lenth, 2020)). We used the DHARMa package to examine residuals to check for normality and homogeneity of variance in our models (Hartig, 2021). For all models using proportion data, the proportion of time was logit transformed to stabilize variances, using the formula ln((p + 0.01)/(1-p + 0.01)) where p is the proportion of time and 0.01 was added as there were zeros in the proportion data that cannot be transformed using the logit scale.
For the two-way trials, we assessed the data in four groups: whether the focal female was hecki or acuticauda, and whether the bill color of stimulus males was natural or artificial. We performed linear mixed models to test whether the proportion of time spent with stimulus birds differed depending on stimulus bird bill color, with focal bird ID and trial number as random effects. For the trials with acuticauda focal females and natural bill colors, each stimulus and each focal bird was only used in one trial, so a linear model was applied instead (no random effects). In trials with yellow focal females and natural stimulus bill colors there are two extreme values, but excluding these points produces the same results, so we report results with the full dataset. As there were fewer manipulated trials, we calculated the power they had to detect any effect identified in the natural trials. We analyzed the four-way trials using linear models in a similar fashion to the two-way trials (details in the Supporting Information). To test for evidence of biased inheritance of bill color in F 1 hybrids, we ran a linear model with the independent variable as H3 (hue) and sex and cross (and their interaction) as dependent variables.
When examining the dominance of genes underlying bill color, we found that the bill color hue (H3) of F 1 hybrids was significantly affected by the interaction between sex and cross direction (F = 18.17, Df = 3, p < 0.001): F 1 females with an acuticauda father and a hecki mother had significantly yellower bills than any of the other groups (p < 0.005 for all; Figure 4; Table A1). There was no difference between males of either cross direction and females with a hecki father and an acuticauda mother (p > 0.99 for all; Figure 4; Table A1).

| DISCUSS ION
In this study, we used behavioral assays to evaluate mate choice based on bill coloration in both subspecies of the long-tailed finch (Poephila acuticauda). We found no evidence for a common female preference for yellow billed individuals across both subspecies that could drive the observed introgression of yellow bill color in the wild.
Our results instead suggest that females have a weak assortative preference for their own subspecies, whereby acuticauda females prefer naturally yellow-billed males and hecki females prefer naturally red-billed males. However, this assortative preference weakens or disappears when females are exposed to two males of their own subspecies with bills manipulated to be red or yellow, suggesting that the assortative preference is multimodal or based on another trait that differs between the subspecies (e.g., song). A potential source of the assortative mate preference observed in this study is sexual imprinting, whereby offspring develop their mate preferences by learning during a short sensitive period, based on the phenotypes of the parents that reared them (Brodin & Haas, 2006;Clayton, 1993;Irwin & Price, 1999;Lorenz, 1937). Sexual imprinting has widespread documentation in birds and has received considerable support as a driver of mate preferences in the Estrildid finches (Cate, ten, & Vos, 1999;Clayton, 1990;Oetting & Bischof, 1996;Witte & Sawka, 2003).
Intriguingly we found that all F 1 birds had bill color hue closer to red hecki bills than yellow acuticauda bills, but that F 1 females with an acuticauda father had significantly yellower bills than other F 1 hybrids. This is consistent with one candidate gene for bill color being located on the Z chromosome (female birds have only one copy of the Z chromosome, that is, are hemizygous) as found in Hooper et al. (2019). As female hybrids with hecki fathers did not have significantly redder bills than any other hybrid group (Figure 4), it suggests that the acuticauda contribution to bill color is at least partially recessive to the hecki contribution on the Z chromosome. The hybrid zone of the European crow provides the best understood case of assortative mating-driven introgression, for which simulation models suggest that upon secondary contact the dominance of dark plumage alleles over light ones resulted in high frequency of dark plumage among hybrids (Metzler et al., 2021). The models predict that assortative mating preference then induced positive frequency-dependent selection on dark morphs, increasing the alleles underpinning dark plumage in the hybrid zone, moving the dark plumage into the light plumage lineage (Knief et al. 2019;Metzler et al., 2021). Another study on assortative-mate preference-driven introgression also performed forward simulations that suggested that the direction of introgression can be variable and stochastic, such that in 33% of their simulations it was the recessive phenotype that introgressed into the dominant phenotype lineage (Semenov et al., 2021). This appears to be the case with the recessive dark head plumage of Motacilla alba personata into M. a. alba (Semenov et al., 2017(Semenov et al., , 2021, and in the present study with the introgression of the recessive yellow bill color. The simulations by Semenov et al. (2021) also suggest that relatively weak mate preference, as observed in the current study (Figure 3), can be enough to contribute to the unidirectional introgression of phenotypic traits (Metzler et al., 2021;Semenov et al., 2021). Developing a better understanding of the genetic architecture underlying bill color is now a priority in the long-tailed finch.
Our dataset suggests that there is assortative mate preference for bill color when focal birds are presented with natural bill-colored males, but less so, or not at all, for trials with manipulated bill color. P. acuticauda females showed the same pattern of assortative preference for manipulated bills, but the result was borderline nonsignificant, potentially due to the much lower statistical power resulting from the small sample size (Figure 3).
P. hecki females showed no pattern of assortative preference for manipulated bills (Figure 3), which may be because the artificial red bill colors were not convincing replicates of the natural red bills, as reflected by the H3 values ( Figure 1). As such it remains possible that the assortative preference observed in trials with natural bill colors is truly based on bill color itself, but that we did not find convincing evidence of this due to either low statistical power and/or the slightly unnatural bill color manipulation (e.g. in the UV component). Alternatively, this discrepancy might instead suggest that the assortative preference observed in these data may actually be due to a combination of traits that differs between F I G U R E 3 Female two-way choice trials suggest an associative preference exists for males of their own subspecies. The proportion of time focal females of subspecies acuticauda (a) or hecki (b) spent with stimulus males of either subspecies with unmanipulated natural bill color (i.e., NATural CONspecifc or HETerospecifc choice) and stimulus males of their own subspecies with manipulated bill color (artificial yellow or red). The number of trials (i.e., focal females) is given beneath the results of each of our four experimental treatment groups.

F I G U R E 4
Bill color of first-generation (F 1 ) hybrids is dependent on hybrid sex and the direction of hybrid cross. Variation in bill color hue (H3) is shown for long-tailed finches from subspecies acuticauda and hecki (results from both sexes) and their F 1 hybrids grouped by the direction of hybrid cross (hecki female x acuticauda male or acuticauda female × hecki male) and sex (female or male). All F 1 hybrids have intermediately 'orange' colored bills, however, F 1 hybrid females with acuticauda fathers have significantly yellower bills than F 1 hybrid females with hecki fathers or either class of F 1 hybrid male. Different letters indicate statistical significance between treatment groups (p < 0.05; Tukey's HSD test). shown in the closely related zebra finch Taeniopygia guttata, the assay of female choice using this type of approach might be influenced by the behavior of the stimulus bird, which may depend on the subspecies of both male and female involved (Clayton, 1990;Rutstein et al., 2007). This is difficult to address and would require also manipulating the appearance of the focal female.
In conclusion, we find evidence that females of both subspecies of long-tailed finch appear to have an assortative mate preference for males of their own subspecies but did not find evidence for assortative preference based on artificially manipulated bill color alone. We also found that bill color of F 1 hybrid females was sig-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflicts of interest.

F I G U R E A 4
The first two principal components of spectrophotometry data for the natural and manipulated bill colors used in behavioral trials. Spectrophotometry data from the 300-700 nm range were smoothed and normalized before being input into a principal components analysis. The percentage of variance in the data explained by each components is in brackets.

TA B L E A 1
Pairwise contrast among F1 groups (sex and parental cross), given the significant effect of sex*cross on bill color (H3). Note: Focal Col = the bill color (and subspecies) of the choosing bird, Artificial = whether bill colors of the stimulus birds all had their bill colors manipulated (Y) or none of them did (N).

TA B L E A 3
Four-way mate-choice trials considering the proportion of time spent with each stimulus bird, with Trial ID as a random effect. Note: Due to the low sample sizes some additional potentially confounding factors could not be statistically accounted for, including the genetic lineage of stimulus birds, and that stimulus birds were included in variable numbers of trials.
a Note that here there is a significant difference, but driven by a non-preference for particular group/s, and no obvious preference for a particular phenotype. b Note that here the p-value was <0.05 for the main model, but the post hoc tests of all pairwise comparisons found no significant differences between groups.

TA B L E A 4
Results from principal component analysis (PCA) that was run on spectrophotometry data between 300 and 700 nm, including red (R) and yellow (Y), manipulated (Manip) and natural (Nat) bill colors.